PDX Samples Research Articles

The use of whole exome sequencing and murine patient derived xenografts as a method of chemosensitivity testing in sarcoma

BackgroundSoft tissue and bone sarcoma represent a broad spectrum of different pathology and genetic variance. Current chemotherapy regimens are derived from randomised trials and represent empirical treatment. Chemosensitivity testing and whole exome sequencing (WES) may offer personalized chemotherapy treatment based on genetic mutations.MethodsA pilot, prospective, non-randomised control experimental study was conducted. Twelve patients with metastatic bone or soft tissue sarcoma that had failed first line chemotherapy treatment were enrolled for this study. Human tissue taken at surgical biopsy under general anaesthetic was divided between two arms of the trial. Subsections of the tumour were used for WES and the remainder was implanted subcutaneously in immunodeficient mice (PDX). Results of WES were analysed using a bioinformatics pipeline to identify mutations conferring susceptibility to kinase inhibitors and common chemotherapeutic agents. PDX models exhibiting successful growth underwent WES of the tumour and subsequent chemosensitivity testing.ResultsWES was successful in all 12 patients, with successful establishment PDX tumours models in seven patients. WES identified potential actionable therapeutics in all patients. Significant variation in predicted therapeutics was demonstrated between three PDX samples and their matched tumour samples.ConclusionAnalysis of WES of fresh tumour specimens via a bioinformatics pipeline may identify potential actionable chemotherapy agents. Further research into this field may lead to the development of personalized cancer therapy for sarcoma.

Abstract 1891: Annexin A2-binding S100 proteins promote proliferation and cell cycle progression of EGFR positive cancer cells

Abstract (a) Aim: The purpose of this study is to clarify an unknown function of Annexin A2-binding S100A proteins in tumor growth, cell cycle progression and invasion. Annexin A2 is a membrane bound protein originally identified as v-Src substrate, and often found to be overexpressed in many types of cancers. As a multicompartmental protein that orchestrates a spectrum of dynamic membrane-related events, it is associated with actin cytoskeleton control, vesicle formation and intercellular interaction. (b) Methods and Results: During our proteomics analysis using solid tumor cell lines, we identified both Annexin A2 and S100A proteins in cancer stem cell-rich population. We next examined S100A proteins expression in several Head and Neck cancer PDX samples. Immunohistochemical data from in vivo specimens as well as western blots showed that S100A expression abundance was well associated with poor prognosis. Annexin A2 has been indicated to be involved in malignant phenotypes such as invasion and metastasis, and Annexin A2 and S100A form (S100-Annexin A2)x2 dimers, we next examined roles of S100A proteins in cancer, by using shRNA-mediated depletion experiments. (c) New Findings: Depletion of S100A from PDX-derived cancer cells resulted in loss of both in vivo tumor formation and invasion. Furthermore, cell cycle progression was inhibited by the depletion, suggesting its potential role in proliferation controls. Similar to the mutant Ras-driven tumors, EGFR-overexpression increased S100A gene expresssion. (d) Conclusion: These results strongly suggest that increases in S100A plays significant roles in cancer cell proliferation and invasion. Note: This abstract was not presented at the meeting. Citation Format: Naoko Ogama, Ryuuichi Nagashima, Nobuyuki Tanaka. Annexin A2-binding S100 proteins promote proliferation and cell cycle progression of EGFR positive cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1891. doi:10.1158/1538-7445.AM2017-1891

Abstract A31: Proteomic characterization of stromal contribution to tumor using patient-derived xenografts

Abstract Patient-derived xenografts (PDXs) are an important and powerful tool to study many types of cancer and provide valuable insight into potential drug response and development of resistance. Due to the differences in tissue lineage PDXs also allow the dissection of stromal contribution to the tumor that is not easily obtainable from examination of the original tumor itself. We have applied mass-spectrometry enabled proteomics, phosphoproteomics, and RNAseq to compare primary metastasis, and PDXs derived from several metastatic sites from the same original tumor which was a colorectal adenocarcinoma. Comparing our results with a large-scale proteomic characterization of tumors performed under the clinical tumor analysis consortium (CPTAC) shows that the original metastasis, and PDXs from different sites, are significantly similar, regardless of metastasis site. Proteomic profiles of these samples are enriched in proteins expressed in the tissue of origin, rather than the site of metastasis. Pathway analysis from proteomics revealed enrichment in complement cascade, extracellular matrix receptors, and focal adhesion. These were different than the pathways indicated by the transcriptomics, which included cell cycle, focal adhesion, and MAP signaling, but not complement or extracellular matrix. We found in the case of complement and focal adhesion the original metastasis sample was very different from the PDX samples, if we only considered human protein contributions. Considering mouse proteins we found that the mouse stromal contribution complemented the human tumor contribution in the PDXs, aligning the functional analysis in both types of samples. Combining these results with subsequent analysis of driver mutations revealed that the proteomic contribution of stroma was as high as 40%. We have extended these observations to additional pairs of clinical tumor specimens and paired PDXs, including different tumor types. In all cases, there is greater similarity among the same sample type (resected tumor, metastatic lesion, or PDX) than between different sample types from the same patient. Citation Format: Jason E. McDermott, Tao Liu, Vladislav Petyuk, Richard Smith, Karin Rodland. Proteomic characterization of stromal contribution to tumor using patient-derived xenografts. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A31.

Abstract 605: Building better cell models for screening oncology compounds

Abstract The NCI60 screen is a valuable compound screening service offered to researchers for 20+ years. The screen allows the submission of small molecules, biologics and natural products for testing in the NCI60 cell line panel (60 cell lines covering 9 major tumor types). Sophisticated tools such as COMPARE allow stratification of compounds based on pattern of cell line response and these tools elucidated several compounds with differential specificity for a disease type (e.g. breast cancer over melanoma). Since inception the NCI60 screen has been performed in 96-well plates on monolayer cultures of cells. SulfoRhodamine B was used to measure cell viability after a 48h exposure to the compounds at either a single concentration or 5 point concentration response assay. However, recent work has suggested that cells in 3D culture may respond differently to compounds than in 2D. 3D culture systems produce tumor cell spheroids that exhibit features seen in vivo including greater cell-cell contact, and gradients of nutrients and oxygen between the periphery and the center of the spheroid. We sought to optimize the NCI60 cells for use in a 3D spheroid model that would be suitable for screening compounds. By varying NCI60 cell plating density and incubation times, we optimized the formation of spheroids of a given diameter (500um) in Ultra Low Attachment (ULA) plates. Spheroids were imaged using a high content imaging plate reader and classified into 4 categories based on their apparent morphologies. Comparisons were made in drug sensitivity between 2D and 3D model systems using the NCI60 and cells derived from PDX samples. Data on the optimal cell densities of the NCI60 lines used for spheroid formation as well as concentration response effects of select agents compared in 2D and 3D models are presented. These data will allow others to rapidly utilize these 3D models to determine the differential drug sensitivity of select cell types. Funded by NCI Contract No. HHSN261200800001E. Citation Format: David M. Evans, Michael Selby, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Thomas Silvers, Ralph E. Parchment, Beverly Teicher. Building better cell models for screening oncology compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 605.

Diffuse Large B-Cell Lymphoma Patient-Derived Xenograft Models Capture Molecular and Biologic Heterogeneity and Inform Therapy

Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease defined by different transcriptional classifications, associated signaling and survival pathways and additional recurrent genetic alterations. In the cell-of-origin (COO) scheme, DLBCLs subtypes share certain features with normal germinal center B-cells (GCB) and activated B-cells (ABC). In comparison to GCB DLBCLs, ABC tumors have increased baseline NFκB activity and more frequent genetic alterations of NFκB pathway components. DLBCLs with shared functional features are also defined by the consensus clustering classification (CCC) which delineates B-cell receptor (BCR), Oxidative Phosphorylation (OxPhos) and Host Response (HR) tumors. BCR DLBCLs have increased reliance on BCR-signaling and survival pathways and aerobic glycolysis. BCR-dependent DLBCLs with high or low baseline NFκB activity (which largely correspond to ABC or GCB tumors, respectively) have distinct SYK-PI3K-dependent survival pathways and shared sensitivity to proximal BCR pathway inhibitors. Although DLBCLs have infrequent inactivating somatic mutations of TP53, these tumors commonly have copy number alterations (CNAs) of TP53 and genes encoding cell cycle pathway components.Given the clinical and molecular heterogeneity of DLBCL, we sought to develop faithful subtype-specific model systems to assess targeted therapies. Fresh tumor biopsies from 27 primary LBCLs were implanted under the renal capsule of immune compromised NSG mice. Nine of 27 tumors were successfully expanded in vivo, serially propagated for > 5 generations and considered stable LBCL PDX models. All models were EBV- and had clonal IgH rearrangements. Morphological and immunohistochemical signatures defined 8 PDX models as DLBCL and 1 as EBV- plasmablastic lymphoma (PBL).All LBCL PDX models were subjected to RNA-Seq and classified with respect to COO and CCC subtypes. Models were also evaluated by whole exome sequencing with a modified bait set which captured coding mutations and selected chromosomal rearrangements. Six of 9 DLBCL PDX models were ABC type. These models exhibited mutations of MYD88 alone or in association with PIM1 or CD79B with other alterations, as reported in primary ABC DLBCLs. The remaining 2 DLBCL PDX models were GCB type, with characteristic alterations of GNA13 and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC. Of note, BCL2 and MYC translocations are known adverse prognostic features of primary GCB DLBCL. Certain PDX models had additional mutations including B2M, MLL2, TNFAIP3, MEF2B and TP53.Only 25% (2/8) of the DLBCL PDX models harbored inactivating TP53 mutations whereas 75% (6/8) of tumors exhibited CNAs of TP53 or its upstream modifier, CDKN2A. These data are consistent with the reported incidence and type of TP53 pathway alterations in primary DLBCLs and contrast sharply with the near-uniform presence of TP53 mutations in DLBCL cell lines.Using the CCC classification, 6/8 DLBCL PDX models (both GCBs and 4 of 6 ABCs) were defined as BCR-subtype and 2 models as non-BCR type. To assess the utility of the DLBCL PDX models for functional analysis of BCR signaling, we first assessed cell surface immunoglobulin (sIg) expression by flow cytometry. All 6 BCR-type DLBCLs expressed sIgM whereas the 2 non-BCR DLBCL models and the PBL model lacked sIg. Next, we treated viable tumor cell suspensions with a selective SYK inhibitor, entospletinib (GS-9973). SYK inhibition significantly decreased the proliferation of all 6 BCR-type DLBCLs, but had no effect on the non-BCR-type DLBCLs or the PBL PDX. Given the distinctive SYK/PI3K-dependent signaling and survival pathways in DLBCLs with low or high baseline NFκB, we also assessed selective apoptotic pathway readouts in entospletinib-treated PDX cell suspensions. SYK inhibition selectively upregulated the pro-apoptotic BH3 family member, HRK, in BCR-dependent GCB-type DLBCL PDX samples and significantly downregulated the anti-apoptotic BCL2 family member, BCL2A1, in BCR- dependent ABC-type DLBCL PDX tumors, effects consistent with those previously observed in primary DLBCL samples.In summary, we have established and molecularly characterized faithful PDX models of DLBCL and PBL and demonstrated their usefulness in evaluating novel BCR pathway inhibitors. DisclosuresRodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Shipp:Gilead: Consultancy; Sanofi: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Patient Derived Xenograft (PDX) Models Recapitulate the Genomic-Driver Composition of Acute Leukemia Samples

Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genomic lesions. However, given recent recognition that multiple genomically distinct sub-clones can exist in AML, there is a risk that there may be selection for sub-clones from the transplanted sample that might not fully represent the patient’s disease.We transplanted 160 (70 T-ALL 56 AML, 32 B-ALL, 2 MLL) patient samples of which 120 engrafted into at least 1 irradiated NSG mouse. 45 AML samples engrafted with a median latency 107+/-41 days. Transplantation of 6 PDX AML samples resulted in immunophenotypically identical disease within 87+/-35 days. 2 MLL samples engrafted in 100% of mice with a median latency of 103+/-13 days. 25 B-ALL samples engrafted with a median latency of 95+/-44 days. Secondary transplantation of 3 PDX B-ALL samples resulted in engraftment of leukemia cells with an identical immunophenotype in 100% of transplanted mice within 52+/-3 days. 48 T-ALL samples engrafted in at least one mouse within 50 days. Secondary transplantation of a single T-ALL PDX sample resulted in 100% engraftment within 31+/-10 days.Genomic DNA and total RNA were isolated from 150 (AML: 16Pt+33PDX; MLL 2Pt+6PDX; B-ALL 17Pt+38PDX; T-ALL 19Pt+19PDX) samples. Adaptor ligated sequencing libraries were captured by solution hybridization using baitsets for 405 cancer-related genes and selected introns for 31 genes frequently rearranged for DNA-seq, and 405 cancer-related and 265 genes frequently rearranged for RNA-seq. All libraries were sequenced averaging >500x for DNA and >6M total pairs for RNA (HiSeq). We detected on average 23+/-12 including a mean 5+/-4 known pathogenic variants such as CDKN2A/B deletion (20/13); FLT3 (SNV & -ITD) and NOTCH (11 ea); WT1 and TP53 (10 ea); NRAS (9); PTPN11 (7); NPM1c, PTEN, and KRAS (6) DNMT3A, IDH1/2, and ASXL1 (5 ea); FBXW7, CEBPA, and TET2 (4 ea); PHF6 and NF1 (3 ea); IKZF1, ATM, and JAK2 (2 ea). Analyses of fusion RNA molecules detected known fusions: MLL-AF4 (4); MLL-AF9 (2), CRLF2-P2RY8, ETV6-RUNX1 or TEL-AML1, PBX1-TCF3 (2 ea); MLL-AF10, MLL-ELL, MLL-EP300, MLL-PTD, BCR-ABL, BCL2-IGK, MYH11-CBFB, along with novel fusions: TCF3-OAZ1, RB1-RCBTB2, PAX5-FLI1, and PAX5-MSI2.The mutations found in the 54 patient samples were consistently identified in the 96 PDX, however some cases showing variation in allele frequency between diagnostic and engrafted samples. Collectively, all 1420 and 288 disease relevant variant allele frequency (VAF) correlated significantly between patient and PDX samples (R2=0.55, R2=0.43), respectively. We then assessed VAF changes from diagnostic to PDX sample as a measure of clonal concordance. Diagnostic and PDX sample were considered discordant if at least one disease relevant VAF demonstrated significant variation between these samples, accounted for small variability of infrequent variances considering SD of sequencing detection. 31 samples were scored as concordant and 23 as discordant which were similarly distributed between disease lineages and did not correlate with diseases status, future relapse or overall survival.Using the same rules we further accessed concordance only between PDX samples in 23 cases when patient samples were transplanted into multiple mice. All 10 groups of PDX samples that were concordant with patient samples were also concordant within the groups. 5 groups of PDX samples that were discordant with patient samples were concordant within groups. 8 groups of PDX samples that were discordant with patient samples were also discordant within their groups. Overall 15 samples produced concordant engraftment in mice and 8 samples produced discordant engraftment. We hypothesized that specific genomic lesions in the 8 groups might underline this discordance. Mutations of FLT3, RAS, TP53, PTPN11 and NOTCH1 correlated with clonal discordance.These findings show that the leukemias that are engrafted in mice mirror the genomic diversity of primary leukemia samples, and that the majority of PDX samples have a genotype similar to that observed in the clinical isolate. More importantly, our data demonstrate the feasibility of developing a large, genetically annotated bank of PDX leukemia models that can be used to test and credential novel therapeutics that target driver mutations in different leukemia subsets. DisclosuresStein:Seattle Genetics, Inc.: Research Funding; Janssen Pharmaceuticals: Consultancy. Wang:Foundation Medicine Inc: Employment. Miller:Foundation Medicine: Employment. Armstrong:Epizyme: Consultancy.

Abstract 206: Depletion of ovarian cancer initiating cells by a monoclonal antibody against ROR1

Abstract ROR1 is a type I orphan-receptor tyrosine-kinase-like surface protein that is expressed during embryogenesis and by various cancer cells, but not on normal adult tissues. Its expression is associated with less-well-differentiated tumors with high metastatic potential (Zhang et al Am J Pathol, 2012, PMCID 3509760; Cui et al Cancer Res, 2013, PMCID 3832210). We evaluated the expression of ROR1 in human ovarian cancers and examined the activity of newly developed anti-ROR1 monoclonal antibodies (mAbs, e.g. UC-961) found to have activity against ROR1-expressing breast cancer cell lines and B-cell leukemias. We examined primary human ovarian cancer cells engrafted into immune-deficient NOD/scid mice (PDX) for human ROR1 expression and for phenotypic and functional characteristics of cancer-stem cells (CSC). In each tumor specimen, we observed different proportions of ROR1-expressing cells. Expression of ROR1 associated with markers of CSC, such as aldehyde dehydrogenase 1 (ALDH1). Moreover, the ROR1+ cells from any given PDX sample had a greater capacity to form spheroids in vitro and to engraft immune-deficient mice than did ROR1-negative cells isolated from the same tumor population. Treatment of ovarian cancer cells with the anti-ROR1 mAb UC-961, but not control IgG, reproducibly impaired their ability to form spheroids, and to engraft into immune-deficient NOD/scid mice. In subsequent studies, we isolated ALDH1+ and ALDH1- cells from the same PDX samples. As noted in prior studies (Ginestier et al Cell Stem Cell, 2007, PMCID 2423808), ALDH1+ cells engrafted more effectively than ALDH1- cells. However, treatment of ovarian cancer cells with the UC-961 mAb abrogated the difference in the capacity of ALDH1+ versus ALDH1- cells to engraft NOD/scid mice. Analysis of the few tumors that did develop in mice engrafted with UC-961-treated, but not control IgG-treated, tumor cells revealed that, compared to the original PDX tumor sample, these tumors had lower proportions of ALDH1+ cells and reduced expression of the polycomb ring finger oncogene, BMI1, and changed the expression level of markers associated with the epithelial-mesenchymal transition (e.g. E-Cadherin and vimentin). Collectively, these studies indicate that expression of ROR1 is associated with CSC and that anti-ROR1 mAbs such as UC-961 can interfere with CSC function and self-renewal, suggesting that such biologics may be useful in the treatment of patients with ovarian cancer. Citation Format: Suping Zhang, Bing Cui, Hsien Lai, Grace Liu, Christina Wu, George Widhopf, Rongrong Wu, Fitzgerlad Lao, Richard Schwab, Dennis Carson, Thomas J. Kipps. Depletion of ovarian cancer initiating cells by a monoclonal antibody against ROR1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 206. doi:10.1158/1538-7445.AM2014-206

Abstract 2373: Deep computational analysis of human and mouse specific next-generation sequencing data generated from PDX specimen

Abstract Patient derived xenograft (PDX) samples are powerful tools to analyze tumor biology and drug sensitivity of primary tumors in animal models. However, sequencing studies on those samples face the challenge of mouse stromal contamination, which varies in each of the explanted tumor specimen. Thus, without applying mouse and human specific filters on sequencing data, species specific genomic alterations and differences in expression are impossible to detect. To allow mouse and human specific transcriptome/genome analyses, we adapted our NGS data analysis pipelines to filter for mouse and human specific reads. By applying the analysis algorithms on hybrid-capture and transcriptome sequencing data from 60 lung cancer PDX samples, we were able to generate a comprehensive overview on tumor specific alterations, including point mutations, deletions and insertions, copy number alterations, gene fusions and isoform specific expression levels, as well as species specific genomic alterations and transcriptome wide expression levels. From 5781 nonsynonymous substitutions which were detected by hybrid-capture sequencing of 333 cancer relevant genes (CAGE-Rx Scanner), 863 human specific nonsynonymous substitutions were detected after filtering for mouse specific variations. The mean sequencing coverage of the CAGE analysis across all targeted bases in all 60 samples was 662x. An integrated analysis of this data set and corresponding transcriptome sequencing data revealed TP53 and KRAS as the most frequently mutated genes which were expressed. As expected, due to the relative high tumor content in all samples (ranging from 30 to 90% with a median of 90% as determined by pathological review), 80% of all mutations called from transcriptome sequencing in an unfiltered fashion were human specific and called after species specific filtering. In addition, the detection of mouse specific transcriptome sequencing reads identified several growth factors expressed by tumor surrounding tissue, which would have been discarded as unmapped reads in the normal analysis setting. Thus, our analysis alorithms not only enable to remove mouse specific false positive single nucleotide variants (SNVs), but also a detailed analysis of mouse specific expression patterns which might strongly influence tumor growth via the tumor microenvironment. Citation Format: Roopika Menon, Petra Schneider, Martin Peifer, Frauke Leenders, Johannes M. Heuckmann. Deep computational analysis of human and mouse specific next-generation sequencing data generated from PDX specimen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2373. doi:10.1158/1538-7445.AM2014-2373

An ex vivo assay of XRT-induced Rad51 foci formation predicts response to PARP-inhibition in ovarian cancer

ObjectiveBRCA-positive ovarian cancer patients derive benefit PARP inhibitors. Approximately 50% of ovarian cancer tumors have homologous recombination (HR) deficiencies and are therefore “BRCA-like,” possibly rendering them sensitive to PARP inhibition. However, no predictive assay exists to identify these patients. We sought to determine if irradiation-induced Rad51 foci formation, a known marker of HR, correlated to PARP inhibitor response in an ovarian cancer model. MethodsOvarian cancer cell lines were exposed to PARP-inhibitor ABT-888 to determine effect on growth. Rad51 protein expression prior to irradiation was determined via Western blot. Cultured cells and patient-derived xenograft tumors (PDX) were irradiated and probed for Rad51 foci. In vivo PDX tumors were treated with ABT-888 and carboplatin; these results were correlated with the ex vivo ionizing radiation assay. ResultsThree of seven cell lines were sensitive to ABT-888. Sensitive lines had the lowest Rad51 foci formation rate after irradiation, indicating functional HR deficiency. Approximately 50% of the PDX samples had decreased Rad51 foci formation. Total Rad51 protein levels were consistently low, suggesting that DNA damage induction is required to characterize HR status. The ex vivo IR assay accurately predicted which PDX models were sensitive to PARP inhibition in vitro and in vivo. ABT-888 alone reduced orthotopic tumor growth by 51% in A2780ip2 cell line, predicted to respond by the ex vivo assay. Three PDX models' response also correlated with the assay. ConclusionsThe ex vivo IR assay correlates with response to PARP inhibition. Analysis of total Rad51 protein is not a reliable substitute.